1. Platelet Function and
Dysfunction
Paul Basciano MD
October 24, 2013

2. Outline
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Thrombopoiesis
Platelet Structure
Platelet Function
Platelet Activation and Aggregation Biology
Assessment of Platelet Function
Inherited Disorders of Platelet Function
Acquired Disorders of Platelet Function
Treatment of Platelet Dysfunction

4. PLATELET PRODUCTION,
STRUCTURE, AND FUNCTION

5. Thrombopoiesis
• Humans produce 1x 10^11 platelets per day
• Can increase 10-20 fold
• Each megakaryocyte can produce 1000
platelets
• The lifespan of a platelet is approximately 10
days
• Platelet production from MK progenitors takes
4-7days
• Most of this time is for the maturation of the
megakaryocyte
• Endomitosis
• Cytoplasmic maturation

6. Thrombopoietin
• TPO is one of the key factors for platelet production
• Produced constitutively by the liver (and kidney)
• Circulating levels are determined by the megakaryocyte and
platelet biomass
• Low platelets more circulating TPO  increased platelet
production
• ITP is an exception to this
• Normal platelets-> low TPO->low platelet production

7. Thrombopoietin in Disease

8. Platelet Structure
• Alpha granules (many): PF4, vWF, fibronectin, thromboglobulin, FV, FXI,
PS, TGFB, PDGF
• Dense granules (3-8/cell): ADP, ATP, serotonin, pyrophosphate, calcium
• Open canalicular system
• Dense tubular system

9. Platelet Functions
• Primary hemostasis—the “platelet plug”
• Secondary hemostasis—the coagulation
cascade
• Vasculature maintenance
• Reservoir for soluble factors

10. Collagen (especially Type I and III)
Fibronectin
Thrombospondin
Laminin
Bound-vWF

11. Endothelial
Damage
Basement
Membrane
Exposures
Platelet Adhesion
Platelet Activation
Phospholipid
Exposure
Coagulation Cascade
(Secondary Hemostasis)
Release of Soluble Factors
Shape Change
Integrin activation
Activation of Other
Platelets
Platelet Aggregation

12. Adhesion Phase: vWF
GP Ib/IX/V Complex
• Major receptor modulating interaction with vWF
• GP Ibα is essential for arterial thrombus formation (Fab
development)
• No inherent signaling ability (no coupling to G proteins,
no TK activity)
• Found in lipid rafts in association with other GPs
• Defects define Bernard Soulier Syndrome
• Macrothrombocytopenia
• No response to ristocetin on aggregometry

13. AdhesionPhase: Collagen
GP VI
• Low-affinity but high impact for binding to collagen
• Signals through Src kinases, as well as association with
FcRg dimer which signals through ITAMs
• Ab in development blocks thrombosis but does not
prolong bleeding time
GP Ia/IIa
No specific human disease has been identified with a
defect in either

15. Platelet Activation:
Granule Secretion and Integrin Activation
• Initial binding leads to the release of agonists from the first-responding
platelets which in turn leads to recruitment and activation of other near-by
platelets
• ADP
• TxA2
• Epinephrine
• Thrombin
• All lead to the activation of GP IIb/IIIa (αIIb/β3 integrin) via inside-out signaling
• Also lead to more granule secretion->feed forward mechanism
• Activation of GPIIb/IIIa allows it to bind to fibrinogen (and vWF), to cross link
platelets and allow the thrombus to form

16. Platelet Activation: Outside-In and
Inside-Out signaling

17. Granule Secretion/GPCRs:
ADP and P2Y1 and P2Y12 Receptors
• Released from platelet dense granules and from RBCs
• Interacts with the GPCRs
• Leads to Ca elevation, TxA2 synthesis, protein
phosphorylation, and shape change, granule secretion,
activation of GPIIb/IIIa and aggregation
• P2Y12 is more important
• Few patients identified with dysfunction and bleeding
diathesis
• Major target of thienopyridine drugs
• P2Y1 is necessary for full signaling and is a potential drug target

18. GPCRs:
Thromboxane A2 and Tpa receptor
• The product of COX and TxA2 synthase enzymes using
arachadonic acid (AA) as a precursor
• Freely diffuses across plasma membrane
• High activity during aggregation when platelets are
closely apposed
• Causes shape change, phospholipid hydrolysis, Ca
mobilization, secretion, and aggregation via the TPa
receptor
• Aspirin inhibits COX-1 irreversibly

19. GPCRs:
Epinephrine and α2A Receptor
• Weak agonist—cannot induce shape change or activate PLC
• Reduces levels of cAMP within the platelet

20. GPCRs:
Thrombin and PAR receptors
• Likely the most potent platelet activator
• Acts via PAR-1 and PAR-4 (protease activated receptors)
• The ligand is tethered to the receptor but hidden until it is
cleaved
Nature 407, 258-264(14 September 2000)
• Also Ib/IX/V complex
• Impaired thrombin responsiveness in BSS—Ib/IX possibly localizes
thrombin to the PARs
• PAR-1 is likely the primary mediator, but PAR4 is necessary for full
activation
• No clinical examples of PAR deficiencies have been described

21. Adhesion
Integrin Activation and
Platelet Aggregation
Secretion
GPCRs activation
Ca++, PKC
Fibrinogen
GP IIb/IIIa (αIIBβIII)

22. ASSESSMENT OF PLATELET
FUNCTION

23. Evaluation of Platelet Function
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Peripheral Blood Smear
Bleeding Time
PFA-100
Platelet aggregometry and secretion
Thromboelastography

24. Peripheral Blood Smear
• Keys to look for:
– Number of platelets
– Size of platelets
– Granulations within platelets
– WBC inclusions

25. Bleeding Time
• Controversial, and mainly abandoned
• Invasive, difficult to standardize
• Requires dedicated technician to perform test
over a relatively long period of time on one
patient

26. PFA-100
Epi or ADP
collagen
collagen
• Rapid, automated, general
assessment of platelet and overall
hemostatic function
– Replacing bleeding time
• Citrated whole blood passed
through aperture and time to
closure with platelet plug is
measured
– Collagen with either epinephrine
or ADP
• High negative predictive value;
low specificity

27. PFA-100
• Influenced by many factors:
– Hematocrit
– Platelet count
– Blood group
– Timing and processing

29. Platelet Aggregometry
• Usually performed on platelet-rich plasma
• Measures transmission of light through the
solution full of platelets
– As platelets aggregate, light transmission
increases
• Uses a panel of agonists to determine specific
defect
• Not well-standardized

31. Aggregometry
• Weak agonists:
– ADP and epinephrine: biphasic platelet aggregation
• Strong agonists
– Collagen, TRAP (thrombin), arachadonic acid TXA2
• (Ristocetin: Agglutination)
• Can also measure secretion of ATP and ADP

32. SPECIFIC PLATELET DISORDERS

33. Inherited Platelet Dysfunction

34. Bernard-Soulier Syndrome
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Reduced/lack of GP Ib/IX
Rare, autosomal recessive
Moderate thrombocytopenia
Large platelets
Prolonged bleeding time
Lack of response to ristocetin on platelet
aggregometry (agglutination)
– All other agonists are normal
• ITP vs BSS
• Can diagnose via flow cytometry

35. Glanzmann Thombasthenia
• Qualitative and/or quantitative abnormality of GP
IIb/IIIa
• More severe mucocutaneous bleeding than other
platelet disorders
• Rare, autosomal recessive
• No aggregation response to any agonists except
ristocetin
• Normal secretion to thrombin, but reduced to
weak agonists
• Can diagnose via flow cytometry

36. Storage Pool Disorders:
Dense Granules
• 3-8 dense granules per platelet; will not see
problems on light microscopy
• Contain ADP, ATP, serotonin, Ca, pyrophosphate
• Moderate bleeding diathesis
• Second wave of aggregation to ADP and Epi is lost
• Reduced collagen response
• Seen in association with Hermansky-Pudlak, ChediakHigashi, and Wiskott Aldrich, TAR, and Griscelli
syndromes

37. Hermansky-Pudlak
• Rare autosomal disorder
• Largest concentration in
Puerto Rico
• Occulocutaneous albinism,
congenital nystagmus,
decreased visual acuity
• Also granulomatous colitis
and pulmonary fibrosis

38. Chediak-Higashi
• Occulocutaneous
albinism
• Immune deficiency
• Cytotoxic T and NK cell
dysfunction
• Neurologic symptoms
• Cytoplasmic inclusions
• LYST gene on
chromosome 1
ASH Image Bank

39. Storage Pools Disorders:
Alpha Granules
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Gray platelet syndrome
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Mild thrombocytopenia
Macrothrombocytopenia
Pale platelets on light microscopy
Variable inheritance
Variable aggregation patterns
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Response to ADP and epi normal
Collage, thrombin, and ADP impaired
– Splenomegaly, fibrosis
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Quebec platelet disorder
– Autosomal dominant
– Delayed bleeding
– Increased proteolysis of alpha granule proteins (elevated platelet urokinase type plasminogen
activator)
– Thrombocytopenia
– Reduced aggregation with epinephrine

40. Signal Transduction and Activation
Abnormalities
• Abnormalities for specific agonist receptors or
abnormalities of the signal transduction
cascade
– G proteins, phospholipase C, calcium mobilization,
pleckstrin phosphorylation, phospholipase C, PKD
– Thromboxane synthesis

41. MYH-9 abnormalities (May-Hegglin)
• MYH-9 mutations (myosin heavy chain)
• Macrothrombocytopenia
• Most commonly described as MayHegglin;
– Fechtner, Epstein, Sebastian Syndromes
are all part of the same spectrum
• Neutrophil inclusions (Dohle bodies)
• Abnormalities in kidneys, ears, heart

42. Wiskott-Aldrich
• Characterized by moderate to severe
thrombocytopenia, with small platelet volumes
• Wide clinical variability:
– susceptibility to infections associated with adaptive
and innate immune deficiency
– eczema
– isolated thrombocytopenia
– X-linked neutropenia (XLN)
• Mutation is WASP—actin remodeling protein
• Platelet volumes are usually 3.5-5fL (normal=710fL)

43. Scott Syndrome
• Defect in platelet pro-coagulant activity
• Failure to move phosphatidylserine to the
outer membrane
• Normal bleeding time and normal platelet
aggregation

44. Bleeding disorder
Low Platelet Count
Small Platelets
Large Platelets
Normal Platelet Count, Normal Size
Very Abnormal
Aggregometry
Normal
Aggregometry
Glanzmann
Wiskott-Aldrich
No Color Issues
Bernard Soulier
Scott
Quebec
Color Issues
Pale Platelets
Pale People
Gray Platelet
Normal Neutrophils
Kind-of adapted from
Williams Hematology,
7th Ed.
Neutrophil Inclusions
Hermansky-Pudlack
Chediak-Higashi

45. Acquired Platelet Defects

46. Aspirin
• Irreversibly inhibits COX-1 and leads to
inhibition of thromboxane A2 production
• The most common antithrombotic in use
• Inhibition with low-dose is complete (50100mg)
– Essential thrombocythemia
• Major bleeding risk is approximately 1% per
year
• Other NSAIDs block COX-1 to a lesser degree

47. Thienopyridines
• Clopidogrel, ticlopidine, and prasagurel
• Work (mainly? in part?) by blocking the ADP
receptor
• Multiple methods to asses effect:
aggregometry, PFA-100 (ADP), Verify-Now
– All have significant variability in detection of
‘resistance’ and questionable clinical utility

48. GP IIb/IIIa inhibitors
• Abciximab, eptifibatide, tirofiban
• Complete thrombasthenic state (Glanzman’s
thrombasthenia)
• High incidence of bleeding complications
• May also cause acute thrombocytopenia
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Rapid onset, 30min to one day
Variable recovery
May be severe
May also induce platelet clumping/
pseudothrombocytopenia

49. Other medications
• Penicillins (especially high dose)
– Onset 2-3d, lasts 3-10d after discontinuation
• Cephalosporins
• SSRIs

50. ADP
Epi
Collagen Ristocetan
Bernard Soulier
Glanzmann
Storage
Pool/Secretion
www.practical-hemostasis.com

51. Aggregometry in Platelet Disorders
Disorder
Ristocetin
ADP
Epinephrine Arachadonic
Acid
Collagen
Bernard
Soulier
---
+++
+++
+++
+++
Glanzmann
+++
---
---
---
---
Storage
Pool/Release
Defect
+
Primary only Primary only
ASA
Primary only
Clopidogrel
---
Primary only
-----
---

52. Treatment
• DDAVP for mild bleeding diathesis, uremic
• Platelet transfusions
– Allo-immunization against absent GP receptors
may occur
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Estrogens
Amicar
Novo7 (Esp. Glanzmann)
TPO agents (Bernard Soulier, MYH-9, ?others)